The invention relates generally to modulating physiological responses to singlet oxygen in a bacterial cells, algae or plant phototrophs. Several sources of singlet oxygen in biological systems, including enzymes such as peroxidases and oxidases, as well as processes such as photosynthesis. Kochevar I, “Singlet oxygen signaling: from intimate to global,” STKE 204:pe7 (2004). In the photosynthetic process, input light energy converts water (H2O) and carbon dioxide (CO2) to oxygen (O2) and sugar. Cellular respiration subsequently converts some of the sugar into chemical energy in the form of ATP. The conversion is associated with chlorophyll, a green pigment common to all photosynthetic cells. Although O2 is a relatively non-reactive chemical, when exposed to high-energy or electron-transferring chemical reactions, it can be converted to highly reactive chemical forms collectively designated as “reactive oxygen species” (ROS). ROS are generally considered toxic to organisms because they oxidize carbohydrates, DNA, lipids and proteins, breaking down normal cellular, membrane and reproductive functions. Ultimately, at toxic ROS levels, a chain reaction of cellular oxidation can result in disease or lethality.
Singlet oxygen (1O2) is an ROS produced as a photosynthetic byproduct. In phototrophs, including plants, light energy excites chlorophyll pigments in the light harvesting complexes to a triplet state. At some frequency, an energy transfer from the excited triple state chlorophyll pigments to ground-state O2 generates 1O2 which, as a strong oxidant, can destroy membrane integrity, abolish biomolecular function, and reduce photochemical activity by inactivating photosynthetic enzymes.
Because excited triplet-state chlorophyll pigments and ground-state oxygen are found in close proximity to one another, many phototrophs exhibit some natural defenses against 1O2. For example, carotenoids, fat-soluble, anti-oxidant pigments found within the photosynthetic apparatus, quench 1O2. Telfer A, “What is β-carotene doing in the photosystem II reaction centre,” Phil. Tans. R. Soc. Lond. 357:1431-1440 (2002). Carotenoids include, but are not limited to, β-carotene, zeaxanthin and tocopherols. If not completely quenched by carotenoids, 1O2 can specifically trigger upregulation of genes that encode proteins involved in the molecular defense against photo-oxidative stress. For example, a network of upregulated plant genes maintains a balance between ROS-scavenging proteins and ROS-producing proteins. Mittler R, “Reactive oxygen gene network of plants,” TRENDS in Plant Sci. 9:490-498 (2004). In bacteria, a set of sigma factors, interchangeable RNA polymerase subunits responsible for recognizing transcriptional promoters, maintain essential housekeeping functions and facilitate host response to specific environmental stresses, including ROS. A constitutively-expressed, principal sigma factor is responsible for transcribing essential housekeeping genes. Other sigma factors, transcriptionally- or post-translationally-activated in response to stresses, recognize promoters upstream of genes involved in the response to stresses. Sigma factors are themselves regulated by anti-sigma factors that bind to a specific sigma factor and inhibit that sigma factor's ability to recognize a promoter.
Activation of sigma factors has been studied, inter alia, in Rhodobacter sphaeroides, a member of the α-subdivision of Proteobacteria and a facultative phototroph. R. sphaeroides is among the most metabolically diverse organisms known, being capable of growth under a wide variety of growth conditions. In addition to being photosynthetic, R. sphaeroides possesses additional energy-acquiring mechanisms including lithotrophy, aerobic respiration and anaerobic respiration. SigmaE (σE), a 19.2 kDa alternative sigma factor encoded by rpoE and related to members of the extra-cytoplasmic function (ECF) subfamily of eubacterial RNA polymerase sigma factors, is increased following environmental stress in R. sphaeroides. σE directs transcription from rpoE P1, a promoter for the rpoEchrR operon, and from cycA P3, a promoter for cytochrome c2. Newman J. et al, “The Rhodobacter sphaeroides ECF sigma factor, σE, and the target promoters cycA P3 and rpoE P1,” J. Mol. Biol. 294:307-320 (1999), incorporated herein by reference as if set forth in its entirety. Basal σE activity, however, is quite low because it is complexed with a zinc-dependent anti-sigma factor, ChrR. ChrR loses its ability to inhibit σE if zinc is removed, or if a zinc-binding domain of the N-terminal domain is removed. Newman J, et al., “The importance of zinc-binding to the function of Rhodobacter sphaeroides ChrR as an anti-sigma factor,” J. Mol. Biol. 313:485-499 (2001), incorporated herein by reference as if set forth in its entirety.
GenBank Accession No. AAB17905 (SEQ ID NO:1), discloses the fuill-length R. sphaeroides ChrR sequence. ChrR with a C38R mutation prevented binding to σE. See Newman et al. (1999), supra. Likewise, ChrR with a C35S or a C38S mutation prevented binding to σE. See Newman et al. (2001), supra. Furthermore, a ChrR with a C187/189S mutation was shown to prevent binding to σE. Id. In addition, ChrR with a H6A mutation, a H31A mutation, a C35A mutation or a C38A mutation cannot bind zinc and ultimately cannot bind σE.
GenBank Accession No. AAB17906 (SEQ ID NO:2) discloses the full-length R. sphaeroides σE sequence. Mutations in region 2.1 (amino acids 22 to 46 of SEQ ID NO:2) of σE alter the interaction between ChrR and σE. Anthony J, et al., “Interactions between the Rhodobacter sphaeroides ECF sigma factor, σE, and its anti-sigma factor, ChrR,” J. Mol. Biol. 341:345-360 (2004), incorporated herein by reference as if set forth in its entirety. In particular, σE with a K38E mutation, a K38R mutation or a M42A mutation were less sensitive to ChrR both in vivo and in vitro.
Because 1O2 affects many organisms (including, but not limited to, bacteria, plants, animals and humans), the components of the biological response to 1O2 find application in medicine, agriculture, biotechnology and bioenergy production systems. Animals and plants use 1O2 to defend against microbial pathogens. Davies M, “Reactive species formed on proteins exposed to singlet oxygen,” Photochem. Photobiol. Sci. 3:17-25 (2004), incorporated herein by reference as if set forth in its entirety. For the foregoing reasons, there is a desire to manipulate physiological responses to 1O2 in animals, bacteria and plants. There are many advantages of studying responses to 1O2 in R. sphaeroides. First, one can control the formation of significant amounts of 1O2. Also, biochemical and genetic systems are available to study the response to 1O2 in vivo and in vitro, including an Affymetrix gene chip (Affymetrix; Santa Clara, Calif.), LC/MS proteomics and computation approaches.